| BackgroundPancreatic cancer is the one of malignant tumors, which the incidence rate is rising. Most patients with pancreatic cancer were treated when their symptoms turned out, even if surgical treatment, its prognosis is still poor. Pancreatic cancer have the feature of vascular invasion and perineural growth, lower rate of early diagnosis, easier metastasis. The differentiation of pancreatic cancer and lymph node metastases directly affects the prognosis of patients, therefore, the Experimental study of the treatment of pancreatic cancer is significant.Chemokine is the superfamily of homologous structure and similar function, which initiates and regulates the migration and location of specific cells in the body, many of which are anti-tumor immunocytes. Secondary lymphoid tissue chemokine (SLC) is the subfamily of CC chemokine, which develop anti-tumor immunity by chemotaxis and mediation among the immunocytes, however which has two-way effect. SLC can promotes tumor cells to secrete the proteolytic enzymes, which speeds up the invasion and metastasis of tumor cells, results in the further development of the tumor.Lentivirus vector is a kind of recombinant retroviral vector, reformed from the lentivirus, which has the higher bio-security and expression efficiency of foreign gene, and has the ability to infect dividing cells and non-dividing cells, has the characteristics of more stable expression in vivo. It will not reinfect other cells after infected target cells, and not use the host cell to generate new virus particles, and it has good security. Lentivirus vector has become the focus in gene therapy.ObjectiveIn this research the human CCL21 gene was cloned and pEASY-T1 simple-CCL21 plasmid was constructed. hCCL21 gene was constructed to the lentivirus vector after sequenced. It was certified that Pancreatic cancer cells line PANC-1 expressing CCR7 highly by Real-time PCR. The biological behavior of PANC-1 transfected by Lentivirus-mediated hCCL21 were observed. And then many intracellular signaling pathway genes were detected by PCR microarray gene after transfection, which investigates the role of hCCL21 transfection for pancreatic cancer.Methods1. Human CCL21 gene was cloned by cloning technology, which connected to the pEASY-T1 simple cloning vector, and then sequenced;2. By recombinant technology, human CCL21 gene was enclosed to lentiviral vector PCDH-CMV-MCS-EF1-copGFP, which was concentrated and purificated after determining the concentration;3. Cell lines with high expression of CCR7 was selected by Real-time PCR from human pancreatic cancer cell lines (AsPC-1, BxPC-3, CFPAC-1, PANC-1, SW1990);4. MOI was determined by pre-test of lentiviral transfection. PANC-1 cell line was transfected by lentivirus-mediated hCCL21, which was divided into the experimental group, negative control group and blank control group, and it was verified by PCR, Western blot. The biological behavior of PANC-1 cell line transfected by lentivirus-mediated hCCL21 was observed.5. RT2ProfilerTMPCR microarray includs 84 genes with tumor invasion and metastasis, angiogenesis, apoptosis, adhesion, cell cycle, and signal transduction. The analysis of result of the PCR microarray and Western blot is helpful for investigating the role of hCCL21 for invasion and metastasis of PANC-1. Statistical analysis was performed by Student's t test or one-way ANOVA using SPSS16.0 statistical software. P<0.05 is considered as significant. Results1. Human CCL21 gene was successfully cloned and constructed into pEASY-T1 simple-CCL21 plasmid. It was showed that the cloned hCCL21 gene is identical with the human genebank after sequenced;2. Lentivirus vector(PCDH-CMV-MCS-EF1-copGFP-CCL21) was successfully constructed, and the mature Lenti-hCCL21 was packaged, and the virus titer of which concentrated to 1.25×108TU/ml;3. PANC-1 was selected from human pancreatic cancer cell lines (AsPC-1, BxPC-3, CFPAC-1, PANC-1, SW1990) as high expression of CCR7;4. Lenti-hCCL21 successfully transfects pancreatic cancer cell line PANC-1, and stably expressing, however, the biological behavior of PANC-1 cells have no significant change;5. The result of RT2ProfilerTMPCR microarray shows that 8 genes were significantly changed, two up-regulated genes, which are ATM, BRCA1; down-regulated genes were AKT1, CASP8, FOS, IL8, ITGA2, JUN; According to different functions of gene, we find that CASP8 gene is related with apoptosis, and AKT1, FOS, JUN genes are related with signal transduction, and ATM, BRCA1 genes cell are related with cell cycle, and gene IL8 is related with angiogenesis, and ITGA2 is related with adhesion, In addition, the variation of MMP-9 gene is less than 2 times, the MMP-9 gene expression of the positive experimental group is up-regulated 1.40 times than the blank control group, 1.31 times than the negative control group, while TIMP1 gene of the positive experimental group is down-regulated 1.01 times and 1.02 times than the blank control group and negative control group. The result of Western blot shows that the expression of MMP-9 is increased in the positive experimental group than the negative control and blank control group.ConclusionsThe human CCL21 gene was successfully cloned by cloning technology. And Lenti-hCCL21 was concentrated by recombinant technology, Lenti-hCCL21 stably transfected pancreatic cancer cell line PANC-1 with high expression of CCR7, but the biological behavior of the positive experimental group had no significant change, while the genes are related with invasion and metastasis, angiogenesis, apoptosis, adhesion, cell cycle, and signal transduction have significant change in the positive experimental group by RT2ProfilerTMPCR microarray, which indicates the lentivirus-mediated hCCL21 results in variety of intracellular signal transduction genes; The up-regulation of MMP-9 gene and down-regulation of TIMP1 gene may enhance invasion and metastasis of PANC-1 transfected Lenti-hCCL21, and speed up the proliferation of tumor cells, which lay the foundation for further study in vivo. |
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